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Derek Lowe's commentary on drug discovery and the pharma industry. An editorially independent blog from the publishers of Science Translational Medicine. All content is Derek’s own, and he does not in any way speak for his employer.

Analytical Chemistry

Stereochemical Mysteries, Solved

Ask a chemist (I’ll do) about optical rotation, and you’ll get a confident answer about how right- and left-handed isomers of chiral compounds will rotate polarized light that shines through a solution of one of them. Ask one of us exactly how it does that, and in 99 cases out of a hundred, you’ll witness a hurried change of the subject or a nervous admission that they have no actual idea. The phenomenon is well-known to us chemists, but the deeper explanation is even worse than NMR; you step off into a deep physics hole (by chemist standards) rather quickly. I can get you up to the point that circularly polarized light is actually chiral itself, because of the way their electric and magnetic fields are rotating, and that this means that chiral molecules will actually change the phase velocity of the two different circular polarizations – they don’t come out of the solution the way that they came in.

Beyond that, don’t ask me. But the consequences of this effect are obvious: a solution of a chiral (“handed”) molecule will rotate polarized light, and the amount of that rotation depends on the wavelength of the light, the concentration of the solution (and its path length), and the intrinsic properties of the chiral molecule itself (which is where even more physics kicks in). If you control for those other variables, you can use the optical rotation value of a pure substance as a distinctive identifiable property.

Well, sometimes. As this paper shows, the situation can be pretty messy. Sometimes you have natural products whose optical rotations are reported as varying quite a bit, and when that happens, you know that there’s almost certainly a chiral impurity in there with an intrinsically greater ability to rotate light. And since there’s no way to figure structural information from the actual rotation data itself (sign or magnitude), there can be some open questions. The paper’s from a group at Merck, who have previously shown that you can use calculated VCD (vibrational circular dichroism) spectra to assign absolute chirality. That’s another one of those ideas that formerly was too computationally expensive to do routinely, but has come within reach, and it’s been rewriting some structural assignments the last few years.

And now it’s the turn of frondosin B, and about time. That compound has been synthesized several times, but the absolute chirality has been very much open for debate. The R and S forms have been variously described as having positive and negative optical rotations over the years, with different syntheses flatly disagreeing on which is which (note – this new work apparently invalidates some of the reasoning in that reference!) And these are folks who know what they’re doing (Danishefsky, Trauner, Ovaska, Macmillan, Wright). So what’s going on?

The Merck group calculated the most likely conformers of the molecule and then predicted the IR spectrum that one should obtain. Once that checked out, they moved on to the VCD spectrum. I have never done any VCD work, but it appears to be something of a pain, especially for natural products work. You really need five or ten milligrams of the material, because the signal is very faint, and that’s often just too much material demand for such rare compounds. In addition, the solution has to be quite concentrated, which can be a problem even if you do have enough material on hand. This paper also resorted to ECD, electronic circular dichroism, for which the signal/noise is apparently better (although you have fewer bands to work with). Their calculations showed that the R compound should have a positive sign of rotation.

When Danishefsky and his group synthesized frondosin B, they targeted the R enantiomer and indeed got a small positive optical rotation. That was a pretty close match to the reported value for the natural product itself, so they assigned it as (+)R. But then the Trauner group also synthesized the R compound and got a negative rotation of almost exactly the same magnitude, and they concluded that the natural product was thus (+)S. Trauner thought that Danishefsky’s synthesis had inadvertently flipped a chiral center, while later work (which lined up more with Danishefsky) made people think that it was Trauner’s synthesis that had mistakenly inverted it.

In order to get material to work with, the Merck group painstakingly recreated the syntheses, and found that the problem is not a stereochemical inversion, but an impurity that’s formed afterwards, in a second methylation step. This has the same mass and almost the identical retention time under most chromatographic conditions (which is the real nightmare possibility for anyone doing purification work). The O-demethylation step that is the final step in some of the reported syntheses is very hard on the enantiopurity, as it turns out, but it’s the impurity that causes the main mischief: it has the opposite sign of the natural product, and rotates polarized light ten times more strongly (+13 degrees compared to -155 degrees). The impurity ends up with its second methyl group on a completely different carbon, because of an allyl cation that forms during the reaction (see the paper, which is open-access, for more details). So you end up with the same mass, and extremely similar chromatographic behavior, but with a different structure that gives a completely different optical rotation. As I said, a real nightmare.

So there was no unexpected stereochemical inversion in any of the published syntheses. And it turns out that Danishefsky was correct – the natural product is indeed (+)R. The Trauner synthesis actually did prepare the R compound, as it was designed to, but it was contaminated with enough of the highly-rotating impurity to give a very plausible opposite conclusion. A small but nagging mystery has been solved – and another analytical technique has proven its merit by doing so.

30 comments on “Stereochemical Mysteries, Solved”

Note that the size of the molecules doing the rotating is 1000 fold smaller than the wavelength of the light they are rotating. Could just one molecule do it? Physicists have become pretty good at sending electrons one at a time through the double slit in experiments (spoiler: they still produce an interference pattern), so could this be done with single molecules and single photons?

“The phenomenon is well-known to us chemists, but the deeper explanation is even worse than NMR; you step off into a deep physics hole (by chemist standartds) rather quickly.”

I’ve been working in the field of chiral materials for 3.5 years (with a focus on chiroptical effects) and it’s astonishing how little is understood of some fairly fundamental processes (CISS effect or eMChA, anyone?). I blame a cultural barrier – most physicists don’t really understand chirality (they treat everything as “polarization”, whether chiral or not) while most chemists think that chirality only shows itself when two chiral molecules form a diastereomeric interaction. But the symmetry-breaking of chirality leads to so much more that we don’t fully understand yet!

The last 5-10 years have shown some extremely exciting results, so more and more people are looking into using chirality for cool applications that weren’t traditionally associated with it, e.g. over-potential-free water oxidation. If you’d like to know more, I’d like to shamelessly plug my recent review on this (free to read link in the title).

Speaking of stereochemistry, are you aware that Kurt Mislow died on October 5, age 94? Please allow me a personal reminiscence.

As a high-school kid, age 15, my mentor for my Westinghouse science project was Don Slocum, then a grad student with Kurt. Several of my friends were also in the program. We had learned organic chemistry from Morrison and Boyd (first edition!) the prior summer at an NSF-sponsored program. During the school year, we sat in on Kurt’s graduate-level organic chemistry lectures.

I went to grad school at Princeton because I expected to become an organic chemist and work with Kurt. But when I got there I decided to go a different route, because when I spoke to Walter Kauzmann a new world opened up.

One more earlier recollection. One winter day, Don took his young charges down to the Chemists Club on 45th St. to hear Ron Breslow speak. Given the time frame (1962-3), it was probably about cyclopropenium, but I don’t actually recall.

You may also be aware that Ron also died recently, just five days after Gilbert Stork, who was memorialized here. When I was at Barnard and Columbia, Ron was, I believe, aware of this history, and he was always kind to m.

I wonder if the impurity could have been spotted by tandem mass spectrometry techniques. Especially if the late-stage methylation had been done with 13C-methyl or CD3-methyl groups. (Of course, with only one apparent chromatographic peak at the right m/z for the parent ion, I’d probably think everything was hunky-dory too, and not even try the analysis either.)

If frondosin B and its isomeric impurity coelute, then the tandem mass spectrum would be a composite of the relative contributions of each. Astute and experienced mass spectrometristists might note the anomalous presence of one or more fragment ions that cannot be assigned to the expected structure (if such ions are, indeed, present and relatively abundant) but many might attribute these to a gas-phase rearrangement, which actually may occur. Of course, if the compound and the impurity are separated chromatographically, the respective MS/MS spectra may show telltale differences. Unfortunately the article and the supplemental material do not include any MS data.

– “This has the same mass and almost the identical retention time under most chromatographic conditions (which is the real nightmare possibility for anyone doing purification work).”
They should’ve seen this impurity on NMR, shouldn’t they? Different Me singlets must be quite visible.

NMR (especially 13C NMR) would distinguish them, but you’re limited by S/N. Because the impurity has 100x the specific rotation, a 2% impurity would dominate the rotation. But 50:1 S/N might be hard to get on a small sample.

“The discrepancy in the optical rotation (OR) values obtained in previous studies can be attributed to an undetected minor impurity (ca. 7%) that arose unexpectedly in a key step late in the synthesis.“

7%! How!? That’s a sizeable amount, and probably above the S/N threshold that would really preclude its detection…

One of the great (and disheartening) lessons I learned my first couple months after joining a process chemistry group was that more than half my time in graduate school was wasted because we didn’t have good quantitative analytical chemistry training or tools as they related to organic synthesis.

It’s a pleasure nowadays to work closely with analytical chemists to solve some difficult organic chemistry problems armed with our combined talents and modern instrumentation.

Not from Process, but from basic R&D: When you point out an analytical error and offer to provide better data using improved and approved techniques, you and your suggestions can be dismissed. “Better data” can undermine a beautiful theory and a handful of publications. To some, better data is a bad thing.

Great article that really highlights the importance of analytical chemists and the value that they can add to synthetic endeavours.

A relatively quick and simple experiment here would be a peak purity determination on the lc main peak using the electronic spectra. The impurity is isobaric so ms might not reveal much, but the conjugation differences are marked and you might expect quite different uv spectra that can be picked up in lc software such as chemstation.

That assumes perfect coelution. If the coelution is just partial, then there would be clues that something’s not right from the peak shape, which would lead to an investigation of column efficiency, column fitting and system extra-column volume and dispersion befire confirming that, yes, there’s something else here!

Its tough for lab chemists and PhD students though: some of the above could be a master’s project in their own right and would be a distraction from making more molecules!

There is a company (EC^2) in Belgium that routinely offers the determination of absolute configuration with a combination of VCD and computations. They first use molecular mechanics to screen the conformation space, then select the most stable candidates and run DFT calculations of the VCD spectra. The simulated spectra are then compared with the measured samples. Customers provide the samples and get the assignment in return.
The real power of VCD lies in the fact that the conformation can be determined in solution. Measurement can be a pain, but it is not that bad normally. The acquisition time can be quite long compared with FT-IR spectra.

Thank you for mentioning EC2. EC2 stands for ‘European Center for Chirality’ and is a collaboration between BioTools (the company that commercialized technique of VCD) and two Universities – University of Antwerp and University of Ghent. We indeed provide many services on VCD and ROA – please contact us at ‘info@btools.com’

Completely unrelated to the post but id just like to thank Derek for this blog series. Im a fresh undergrad and ive just started organic labs. Your How not to do it section saved me quite a lot of embarrassment in those first labs (good old acid to water) and all your articles are written in a lovely accessible way. Thanks

@Anon is correct on the use of lenses to decrease the beam size and thus smaller sample cell and smaller amount needed for measurement of VCD.

@dereklowe – Thank your this beautiful write-up. However VCD is no longer a ‘pain’ to measure – it is in fact used by ALL major pharmaceutical companies (and FDA), with over at least 7000 structures solved in the past 10 years or so including over 100 beautiful examples on natural products. You can find some of these papers on our website – http://www.btools.com

To my understand from my grad school, chiral impurity present in sample will effect final OR value. It looks like for me, in this case the small percentage of impurity which is not observed in regular 1H&13CNMR spectra and influenced a lot on OR and mislead all the story.
To my knowledge, methyl gps and their bearing Quaternary carbons in R-8 and RR-10 are highly distinguishable in 13C NMR. If you run 13 C NMR for long time (more scans than usual) or with little high compound to see good high intense carbon (especially Quaternary C) signals, then you will see few shoulder peaks, which would have reveal us the presence of impurity.
May be in Ion Mobility Tandem Mass Spec you can separate these two isobaric compounds.